Using cloned cDNA probes we have isolated the genomic sequences (from the chicken) for the following proteins: alpha skeletal muscle actin, alpha cardiac actin, beta cytoplasmic actin, myosin light chains 1 and 3, vimentin (a major intermediate filament protein), pyruvate kinase, and GAPDH (glyceraldehyde phosphate dehydrogenase). These have been defined by DNA sequence analysis. The various actin genes have been subcloned into the eukaryotic expression vector, pRSV-CAT, in order to study the developmental regulation of these genes after transfection (calcium phosphate precipitation) into the C2 mouse muscle cell line. Probes specific for the various chicken actin genes have been used to monitor expression during differentiation in vitro. In vivo transcription of the vimentin gene, a single copy gene, yields two distinct mRNA transcripts by Northern analysis. The coding potential and formation of these transcripts has been determined. The myosin light chains 1 and 3 are encoded by a single gene. The structure and regulation of this expression is under study. The mouse histone H4 gene is expressed in a cell-cycle dependent fashion. The structure of the gene has been altered to determine, in transfection studies with L-cells and C2 cells, which regions of the gene confer this regulation. Pyruvate kinase undergoes an isoform shift during myogenesis. The structure and regulation of the pyruvate kinase gene is under investigation. The three Acantha amoeba myosin polypeptides have been synthesized in vitro in preparation for the isolation of the three genes. The genes will be used in structural comparison of the myosins.